Proteasomes are giant molecular complexes that degrade intracellular proteins in a regulated manner. They are composed of two subassemblies- a 19S regulatory particle (RP) and a proteolytic 20S catalytic particle (CP). The objective of this application is to understand functionally relevant proteasome structural dynamics. Recent findings from several labs provide subnanometer resolution structures of the proteasome. These beautiful models, however, are incomplete. First, substrate is absent. In the first Specific Aim it will be determined how protein substrates engage the proteasome. This Aim will investigate the mode of interaction between substrate and three key proteasome sites of action: substrate binding, extended polypeptide undergoing active translocation and the constriction against which a folded domain unravels. Second, the present models are static. In the second Specific Aim various biochemical approaches will be used to trap substrate engagement with proteasome in a set of specific conformational states associated with distinct functional states. A widely accepted yet untested paradigm is that protein unfolding and translocation is coupled with large-scale conformational changes of the proteasome. This view will be tested by determining structures of proteasomes with substrate processing in specific states. The third Specific Aim will investigate the structural basis of recent biochemical observations demonstrating homotropic allostery within the proteasome CP. Communication within the CP may be important in biochemical regulation and in determining how the cell allocates the CP pool among various forms of classic and hybrid proteasomes. The rationale for structuring this application with two PIs is that accomplishing the scientific goals of this project requires their continuing interaction to exploit and further develop methods that originated in the labs of each of the applicants: Novel cryoEM methods, which enable rapid high-resolution structure determination, and novel proteasome substrates, which enable formation of stable and tunable proteasome-substrate complexes for structural studies. The proposed studies will visualize functionally relevant proteasome complexes at atomic- level resolution. Proteasomes are fueled by ATP binding and hydrolysis and use the energy so derived to move and unfold their substrates. However, little information is available that relates proteasome function to structural dynamics. The long term goal of these investigations is to understand the biomechanics of proteasome action. The proposed research is significant because it is a first step in transforming our understanding of proteasomes from static objects to dynamic players.